Alsin is a Rab5 and Rac1 guanine nucleotide exchange factor

ALS2 is the gene mutated in a recessive juvenile form of amyotrophic lateral sclerosis (ALS2). ALS2 encodes a large protein termed alsin, which contains a number of predicted cell signaling and protein trafficking sequence motifs. To gain insight into the overall function of alsin and to begin to evaluate its role in motor neuron maintenance, we examined the subcellular localization of alsin and the biochemical activities associated with its individual subdomains. We found that the Vps9p domain of alsin has Rab5 guanine nucleotide exchange activity. In addition, alsin interacted specifically with and acted as a guanine nucleotide exchange factor for Rac1. Immunofluorescence and fractionation experiments in both fibroblasts and neurons revealed that alsin is a cytosolic protein, with a significant portion associated with small, punctate membrane structures. Many of these membrane structures also contained Rab5 or Rac1. Upon overexpression of full-length alsin, the overexpressed material was largely cytosolic, indicating that the association with membrane structures could be saturated. We also found that alsin was present in membrane ruffles and lamellipodia. These data suggest that alsin is involved in membrane transport events, potentially linking endocytic processes and actin cytoskeleton remodeling.     

Characterization of RIN3 as a guanine nucleotide exchange factor for the Rab5 subfamily GTPase Rab31

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.     

EspM2 is a RhoA guanine nucleotide exchange factor

We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide‐free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H‐Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF.     

Asef is a Cdc42-specific guanine nucleotide exchange factor

Asef is a member of the Dbl-family of guanine nucleotide exchange factors (GEFs) with a proposed specificity for the small GTPase Rac1. Here we investigated the specificity and regulation of Asef by measuring its GEF activity in vitro and observed hardly any activity towards Rac1, Rac2 and Rac3, or RhoA and TC10. In contrast, various purified Asef protein fragments catalyzed the nucleotide exchange reaction of Cdc42. The Cdc42GEF activity of the Dbl homology (DH) domain of Asef was significantly higher in the presence of the pleckstrin homology (PH) domain. Our data strongly suggest that Asef is a canonical Cdc42GEF, which employs its PH domain to efficiently stabilize its autoinhibited state, but also to facilitate nucleotide exchange activity of the DH domain after its activation by upstream signals.     

GRAB: a physiologic guanine nucleotide exchange factor for Rab3A, which interacts with inositol hexakisphosphate kinase

Diphosphoinositol-pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate (InsP8) possess pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by recently identified InsP6 kinases designated InsP6K1 and InsP6K2. We now report the identification, cloning, and characterization of a novel protein, GRAB (guanine nucleotide exchange factor for Rab3A), which interacts with both InsP6K1 and Rab3A, a Ras-like GTPase that regulates synaptic vesicle exocytosis. GRAB is a physiologic GEF (guanine nucleotide exchange factor) for Rab3A. Consistent with a role of Rab3A in synaptic vesicle exocytosis, GRAB regulates depolarization-induced release of dopamine from PC12 cells and nicotinic agonist-induced hGH release from bovine adrenal chromaffin cells. The association of InsP6K1 with GRAB fits with a role for InsP7 in vesicle exocytosis.     

A posttermination ribosomal complex is the guanine nucleotide exchange factor for peptide release factor RF3

The mechanism by which peptide release factor RF3 recycles RF1 and RF2 has been clarified and incorporated in a complete scheme for translation termination. Free RF3 is in vivo stably bound to GDP, and ribosomes in complex with RF1 or RF2 act as guanine nucleotide exchange factors (GEF). Hydrolysis of peptidyl-tRNA by RF1 or RF2 allows GTP binding to RF3 on the ribosome. This induces an RF3 conformation with high affinity for ribosomes and leads to rapid dissociation of RF1 or RF2. Dissociation of RF3 from the ribosome requires GTP hydrolysis. Our data suggest that RF3 and its eukaryotic counterpart, eRF3, have mechanistic principles in common.     

Rin-like, a novel regulator of endocytosis, acts as guanine nucleotide exchange factor for Rab5a and Rab22

RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis.     

Gapex-5, a Rab31 guanine nucleotide exchange factor that regulates Glut4 trafficking in adipocytes

Insulin stimulates glucose uptake by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly; the mechanism underlying this process remains uncertain. Using the TC10-interacting protein CIP4 as bait in a yeast two-hybrid screen, we cloned a RasGAP and VPS9 domain-containing protein, Gapex-5/RME-6. The VPS9 domain is a guanine nucleotide exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trans-Golgi network (TGN)-to-endosome trafficking. Overexpression of Rab31 blocks insulin-stimulated Glut4 translocation, whereas knockdown of Rab31 potentiates insulin-stimulated Glut4 translocation and glucose uptake. Gapex-5 is predominantly cytosolic in untreated cells; its overexpression promotes intracellular retention of Glut4 in adipocytes. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, thus reducing Rab31 activity and permitting Glut4 vesicles to translocate to the cell surface, where Glut4 docks and fuses to transport glucose into the cell.     

RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin

The Rab family of small guanosine triphosphatases (GTPases) plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs). Ras and Rab interactor (or Ras interaction/interference)-like (RINL), which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM) domain-containing (Anks) protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.     

Very-KIND is a novel nervous system specific guanine nucleotide exchange factor for Ras GTPases

The kinase non-catalytic c-lobe domain (KIND) evolved from the catalytic protein kinase fold into a potential protein interaction module for signalling proteins. Spir family actin organizers and the non-receptor phosphatase type 13 (PTP type 13) encode a KIND domain in the very N-terminal parts of the proteins. Here we report the characterization and cloning of a third member of the KIND protein family, which we have named very-KIND (VKIND) because of its two KIND domains. Like the other members of the protein family, VKIND has a KIND domain at the N-terminus. A second KIND domain is located in the central part of the protein. The C-terminal half encodes a guanine nucleotide exchange factor motif for Ras-like GTPases (RasGEF) and a RasGEF N-terminal module (RasGEFN). There is only one VKIND gene in the mammalian genomes and up to now we have found the gene only in vertebrates. During mouse embryogenesis the VKIND gene was specifically expressed in the developing nervous system. In adult mice Northern hybridizations revealed high expression only in brain. Low expression could be detected in ovary. In situ hybridizations showed a specific expression of VKIND in neuronal cells of the granular and Purkinje cell layers of the cerebellum.     

Collagen phagocytosis is regulated by the guanine nucleotide exchange factor Vav2

Collagen phagocytosis is a crucial alpha2beta1-integrin-dependent process that mediates extracellular matrix remodeling by fibroblasts. We showed previously that after initial contact with collagen, activated Rac1 accelerates collagen phagocytosis but the Rac guanine nucleotide exchange factors (GEFs) that regulate Rac are not defined. We examined here the GEFs that regulate collagen phagocytosis in mouse fibroblasts. Collagen binding enhanced Rac1 activity (5-20 min) but not Cdc42 or RhoA activity. Analysis of collagen bead-associated proteins showed enrichment with Vav2, which correlated temporally with increased Rac1 activity. Knockdown of Vav2 prevented Rac activation, recruitment of Rac1 to collagen bead binding sites, and collagen bead binding, but knockdown of Sos-1 or beta-Pix had no effect on Rac activation or collagen binding. Vav2 was associated with the nucleotide-free Rac1 mutant (G15ARac1) after collagen binding. Collagen bead binding promoted phosphorylation of Vav2, which temporally correlated with Rac1 activation and which required Src kinase activity. Blockage of Src activity prevented collagen bead-induced Rac activation and collagen bead binding. Collectively these data indicate that Vav2 regulates the Rac1 activity associated with the binding step of collagen phagocytosis.     

SmgGDS is a guanine nucleotide exchange factor that specifically activates RhoA and RhoC

SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.     

RanBP10 is a cytoplasmic guanine nucleotide exchange factor that modulates noncentrosomal microtubules

Microtubule spindle assembly in mitosis is stimulated by Ran.GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including megakaryocytes. In these cells, platelet biogenesis requires massive mobilization of microtubules in the cell periphery, where they form proplatelets, the immediate precursors of platelets, in the apparent absence of centrioles. Here we identify a cytoplasmic Ran-binding protein, RanBP10, as a factor that binds beta-tubulin and associates with megakaryocyte microtubules. Unexpectedly, RanBP10 harbors GEF activity toward Ran. A point mutation in the candidate GEF domain abolishes exchange activity, and our results implicate RanBP10 as a localized cytoplasmic Ran-GEF. RNA interference-mediated loss of RanBP10 in cultured megakaryocytes disrupts microtubule organization. These results lead us to propose that spatiotemporally restricted generation of cytoplasmic Ran.GTP may influence organization of the specialized microtubules required in thrombopoiesis and that RanBP10 might serve as a molecular link between Ran and noncentrosomal microtubules.     

Ric-8B is a GTP-dependent G protein alphas guanine nucleotide exchange factor

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Gα(q) and Gα(s) regulation of neurotransmission, Gα(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and Gα(olf)-dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for Gα(i)/α(q)/α(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a Gα(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be Gα subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed Gα(s) and lesser amounts of Gα(q) and Gα(13). Ric-8BFL stimulated guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding to these subunits and Gα(olf), whereas the Ric-8BΔ9 isoform stimulated Gα(s short) GTPγS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V(max) of Gα(s) steady state GTP hydrolysis and the apparent K(m) values of GTP binding to Gα(s) from ～385 nm to an estimated value of ～42 μM. Directionality of the Ric-8BFL-catalyzed Gα(s) exchange reaction was GTP-dependent. At sub-K(m) GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free Gα(s) tightly, and near-K(m) GTP levels were required to dissociate the Ric-8B·Gα nucleotide-free intermediate to release free Ric-8B and Gα-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce Gα-GTP in cells.     

P-Rex1 is a primary Rac2 guanine nucleotide exchange factor in mouse neutrophils

Leukocyte chemoattractants regulate many leukocyte functions, including leukocyte chemotaxis, via the Rho family of small GTPases that include RhoA, Cdc42, and Rac. Previous work has revealed mechanisms by which chemoattractants regulate RhoA and Cdc42 in mouse neutrophils, but the mechanisms for regulation of Rac remain unclear even though Rac is important for neutrophil functions. Here, we characterized P-Rex1, a Gbetagamma and PIP(3)-regulated guanine nucleotide exchange factor that was initially identified as a Rac activator in response to chemoattractants, for its roles in the regulation of Rac activity and neutrophil functions. We generated a mouse line in which the P-Rex1 gene is disrupted and found that P-Rex1 deficiency did not significantly affect Rac1 activation but diminished Rac2 activation in response to a chemoattractant fMLP in mouse neutrophils. This preference for Rac2 may partially result from the apparent higher affinity of P-Rex1 for Rac2 than for Rac1 because P-Rex1 was more readily immunoprecipitated with Rac2(S17N) than Rac1(S17N). In addition, P-Rex1 deficiency significantly attenuated fMLP-induced F actin formation and superoxide production without affecting LPS- or PMA-induced production. Furthermore, P-Rex1 deficiency caused a chemotactic defect that is primarily attributed to a reduction in the migration rate rather than directionality.     

Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1.

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.     

GrinchGEF--a novel Rho-specific guanine nucleotide exchange factor

RhoGTPases, which are activated by specific guanine nucleotide exchange factors (GEFs), play pivotal roles in several cellular functions. We identified a new RhoGEF (GrinchGEF) containing the typical Dbl homology domain, a putative WD40-like domain, and two predicted transmembrane helices. In contrast to most other RhoGEFs, it exhibits no sequence similarities to known pleckstrin homology domains. GrinchGEF mRNA was highly abundant in skeletal muscle and pancreas. Despite the predicted transmembrane domains, subcellular localization studies revealed a cytosolic distribution. In vitro, GrinchGEF induced the GDP/GTP exchange at RhoA, but not at Rac1 or Cdc42. In intact cells, GrinchGEF induced specifically Rho activation and enhanced RhoA-C-specific downstream effects.     

Insulin-stimulated GLUT4 protein translocation in adipocytes requires the Rab10 guanine nucleotide exchange factor Dennd4C

Insulin-stimulated translocation of the glucose transporter GLUT4 to the cell surface in fat and muscle cells is the basis for insulin-stimulated glucose transport. Studies in adipocytes strongly support the following molecular mechanism for this process. Insulin-elicited phosphorylation of the GTPase-activating protein TBC1D4 (AS160) suppresses its activity toward Rab10 and thereby leads to an increase in the GTP-bound form of Rab10, which in turn triggers movement of vesicles containing GLUT4 to the plasma membrane and their fusion with the membrane. This process is expected to require the participation of a guanine nucleotide exchange factor (GEF) to generate the GTP-bound form of Rab10, but this GEF has not hitherto been identified. The present study identifies Dennd4C, a recently described GEF for Rab10, as the primary GEF required for GLUT4 translocation. Knockdown of Dennd4C markedly inhibited GLUT4 translocation, and ectopic expression of Dennd4C slightly stimulated it. Dennd4C was found in isolated GLUT4 vesicles. This study thus identifies another key component in the machinery of GLUT4 translocation. Moreover, it provides a potential explanation for the moderate association of a variant in the Dennd4C gene with type 2 diabetes.     

New functions for a vertebrate Rho guanine nucleotide exchange factor in ciliated epithelia

Human ARHGEF11, a PDZ-domain-containing Rho guanine nucleotide exchange factor (RhoGEF), has been studied primarily in tissue culture, where it exhibits transforming ability, associates with and modulates the actin cytoskeleton, regulates neurite outgrowth, and mediates activation of Rho in response to stimulation by activated Galpha12/13 or Plexin B1. The fruit fly homolog, RhoGEF2, interacts with heterotrimeric G protein subunits to activate Rho, associates with microtubules, and is required during gastrulation for cell shape changes that mediate epithelial folding. Here, we report functional characterization of a zebrafish homolog of ARHGEF11 that is expressed ubiquitously at blastula and gastrula stages and is enriched in neural tissues and the pronephros during later embryogenesis. Similar to its human homolog, zebrafish Arhgef11 stimulated actin stress fiber formation in cultured cells, whereas overexpression in the embryo of either the zebrafish or human protein impaired gastrulation movements. Loss-of-function experiments utilizing a chromosomal deletion that encompasses the arhgef11 locus, and antisense morpholino oligonucleotides designed to block either translation or splicing, produced embryos with ventrally-curved axes and a number of other phenotypes associated with ciliated epithelia. Arhgef11-deficient embryos often exhibited altered expression of laterality markers, enlarged brain ventricles, kidney cysts, and an excess number of otoliths in the otic vesicles. Although cilia formed and were motile in these embryos, polarized distribution of F-actin and Na(+)/K(+)-ATPase in the pronephric ducts was disturbed. Our studies in zebrafish embryos have identified new, essential roles for this RhoGEF in ciliated epithelia during vertebrate development.